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Fast-scan cyclic voltammetry (FSCV) detection of endogenous octopamine in Drosophila melanogaster ventral nerve cord

机译：快速扫描循环伏安法（FSCV）检测果蝇黑腹腹神经索中的内源性章鱼胺

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摘要

Octopamine is an endogenous biogenic amine neurotransmitter, neurohormone, and neuromodulator in invertebrates, and has functional analogy with norepinephrine in vertebrates. Fast-scan cyclic voltammetry (FSCV) can detect rapid changes in neurotransmitters, but FSCV has not been optimized for octopamine detection in situ. The goal of this study was to characterize octopamine release in the ventral nerve cord of Drosophila larvae for the first time. An FSCV waveform was optimized so that the potential for octopamine oxidation would not be near the switching potential where interferences can occur. Endogenous octopamine release was stimulated by genetically inserting either the ATP sensitive channel, P2X2, or the red-light sensitive channelrhodopsin, CsChrimson, into cells expressing tyrosine decarboxylase (TDC), an octopamine synthesis enzyme. To ensure that release is due to octopamine and not the precursor tyramine, the octopamine synthesis inhibitor disulfiram was applied, and the signal decreased by 80%. Stimulated release was vesicular and a 2 s continuous light stimulation of CsChrimson evoked 0.22 ± 0.03 μM of octopamine release in the larval VNC. Repeated stimulations were stable with 2 or 5 minutes interstimulation times. With pulsed stimulations, the release was dependent on the frequency of applied light pulse. An octopamine transporter has not been identified, and blockers of the dopamine transporter and serotonin transporter had no significant effect on the clearance time of octopamine, suggesting they do not take up octopamine. This study shows that octopamine can be monitored in Drosophila, facilitating future studies of how octopamine release functions in the insect brain.
机译：章鱼胺是无脊椎动物中的内源性生物胺神经递质，神经激素和神经调节剂，在脊椎动物中与去甲肾上腺素具有类似的功能。快速扫描循环伏安法（FSCV）可以检测神经递质的快速变化，但FSCV尚未针对现场的章鱼胺检测进行优化。这项研究的目的是首次表征果蝇幼虫腹神经索中的章鱼胺释放。优化了FSCV波形，以使章鱼胺氧化的电势不会接近发生干扰的开关电势。通过将ATP敏感通道P2X2或红光敏感视紫红质CsChrimson遗传插入表达酪氨酸脱羧酶（TDC）（一种章鱼胺合成酶）的细胞中，可以刺激内源性章鱼胺释放。为确保释放是由于章鱼胺而不是前体酪胺引起的，使用了章鱼胺合成抑制剂双硫仑，信号降低了80％。囊泡刺激释放，持续2 s的CsChrimson光刺激引起幼虫VNC中0.22±0.03μM的章鱼胺释放。重复刺激在2到5分钟的刺激时间内是稳定的。对于脉冲刺激，释放取决于所施加的光脉冲的频率。尚未发现章鱼胺转运蛋白，多巴胺转运蛋白和5-羟色胺转运蛋白的阻滞剂对章鱼胺的清除时间没有明显影响，表明它们不吸收章鱼胺。这项研究表明，可在果蝇中监测章鱼胺，从而有助于进一步研究章鱼胺在昆虫脑中的释放功能。

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